THE CONTRIBUTION OF CLAMS ON TIDAL FLAT PURIFICATION CAPACITY

The function of clams on the decomposition and biodegradability improvement of organic matters in tidal flat sediment were investigated. Moreover, nitrate removal capacity of tidal flat sediments with and without clams was also evaluated. It was found that concentration of DOC in tidal flat sediment with clams was about 1.1-2.1 mg/l higher than that in the sediment without clams. Also NH4-N concentration in the sediment with clams was 0.8-1.8 mg/l higher than that in the sediment without clams. DOC from the sediment with clams was readily biodegradable, while DOC in the sediment without clams was inherent biodegradable substance. Molecular weight of organic matters was distributed from 0.7x102 to 1.7x105 and 0.7x102 to 1.2x106 in the pore water of the sediment with and without clams, respectively. This indicated that clams accelerated the degradation of particulate organic matters and produced DOC, NH4-N and others compounds with higher biodegradability and lower molecular weight. The increase of readily biodegradable compounds as carbon and nutrients sources enhanced microbial growth and accelerated nitrate removal process in tidal flat sediment with clams

Journal of Water and Environment Technology, Vol.2, No.2, 2004 - 83 - THE CONTRIBUTION OF CLAMS ON TIDAL FLAT PURIFICATION CAPACITY Udin Hasanudin 1* , Tadao Kunihiro 2 , Masafumi Fujita 3 , Hong-Ying Hu 4 , Koichi Fujie 1 and Teruaki Suzuki 5 1 Department of Ecological Engineering, Toyohashi University of Technology, Tempaku-cho, Toyohashi 441-8580 Japan (E-mail: udin@fujielab.eco.tut.ac.jp and fujie@eco.tut.ac.jp) 2 Faculty of Environmental and Symbiotic Sciences, Prefectural University of Kumamoto 3-1-100 Tsukide, Kumamoto City 862-8502 (E-mail: tadao92@mwa.biglobe.ne.jp) 3 Department of Civil and Environmental Engineering, University of Yamanashi, 4-3-11 Takeda, Kofu, Yamanashi 400-8511, Japan (E-mail: fujita-m@ccn.yamanashi.ac.jp ) 4 Department of Environmental Science and Engineering, Tsinghua University, Beijing, 100084 China (E- mail: hyhu@tsinghua.edu.cn ) 5 Aichi Fisheries Research Institute, 97, Wakamiya, Miya-cho, Gamagori, 443-0021, Japan (E-mail: teruaki_suzuki@pref.aichi.lg.jp ) ABSTRACT The function of clams on the decomposition and biodegradability improvement of organic matters in tidal flat sediment were investigated. Moreover, nitrate removal capacity of tidal flat sediments with and without clams was also evaluated. It was found that concentration of DOC in tidal flat sediment with clams was about 1.1-2.1 mg/l higher than that in the sediment without clams. Also NH 4 -N concentration in the sediment with clams was 0.8-1.8 mg/l higher than that in the sediment without clams. DOC from the sediment with clams was readily biodegradable, while DOC in the sediment without clams was inherent biodegradable substance. Molecular weight of organic matters was distributed from 0.7x10 2 to 1.7x10 5 and 0.7x10 2 to 1.2x10 6 in the pore water of the sediment with and without clams, respectively. This indicated that clams accelerated the degradation of particulate organic matters and produced DOC, NH 4 -N and others compounds with higher biodegradability and lower molecular weight. The increase of readily biodegradable compounds as carbon and nutrients sources enhanced microbial growth and accelerated nitrate removal process in tidal flat sediment with clams. Keywords: Biodegradability; clams; nitrate removal; organic matters; tidal flat. INTRODUCTION Tidal flat is recognized as important sites for nutrient transformation and sequestration via bio- geochemical cycling (Ogilvie et al., 1997, Magalhaes et al., 2003). The increases of population, urbanization and industrialization enhance nutrient load to environment, including tidal flat. Overload of nutrient to tidal flat area will cause nutrient accumulation and finally the purification functions of tidal flat become decrease. This condition will promote eutrophication and often cause phytoplankton blooms followed by deposition of organic matters and detritus to the seabed sediment (Ueda et al., 2000). The large input of organic matter resulted in anoxia within the sediment, as a consequence of respiration processes, and an enhancement the activities of sulfate- reducing bacteria to produce hydrogen sulfide (Okabe et al., 1998; Graco et al., 2001; Ueda et al., 2000). Oxygen-deficient water further upwelled toward the surface layer, and moved towards the shore, causing a catastrophic impact to the benthic ecosystem of the intertidal flats in the innermost parts of the bay (Ueda et al, 2000). The improvement of tidal flat purification function is necessary to enhance nutrient removal from tidal flat area and prevent eutrophication as an effort of tidal flat conservation. Journal of Water and Environment Technology, Vol.2, No.2, 2004 - 84 - Benthic nutrient regeneration is known as a major driving force in the dynamics of biophilic elements in coastal marine ecosystems. It has been shown that the macrozoobenthos, particularly dense assemblages of bivalves, play a fundamental role in nutrient regeneration processes through their excretory products (Magni et al., 2000). However, uncertainty about the actual contribution of macrozoobenthos to the tidal flat purification capacity may still remain due to insufficient information about the symbiotic function between microorganisms and macrozoobenthos on organic matters decomposition. One of the important macrozoobenthos in tidal flat is clam (Tapes philippinarum). Tapes philippinarum is the major contributor species to the macrozoobenthic biomass on the tidal flat (Magni et al., 2000). Clams as a suspended feeder were recognized to have filtering capabilities by consuming particulate organic matters (POM) and excreting their feces. Clams were efficient at removing particle with has diameter between 3-10 µm (Pfeiffer et al., 1999). Clams excretion activities produced ammonia nitrogen (NH 4 -N), nitrate (NO 3 -N), nitrite (NO 2 -N), total Kjeldahl nitrogen (TKN), phospate (PO 4 -P), and BOD (Magni et al., 2000; Zhu et al., 1999). Thus, the filtering function of clams might produce carbon and nutrient with higher biodegradability. The biodegradability improvement of organic matters is useful to enhance microbial growth and accelerate mineralization process in tidal flat sediment (Hasanudin et al., 2004). The objectives of this research are to investigate the effects of clams on the decomposition and biodegradability improvement of organic matters in tidal flat sediment. Moreover, nitrate removal capacity of tidal flat sediments with and without clams was also studied to clarify the contribution of clams on nutrient removal. MATERIALS AND METHODS Research sites All experimental plots were located in Aichi Fisheries Research Institute, close to Mikawa Bay, Gamagori, Aichi, Japan. The experiment was conducted in two plots of mesocosms of tidal flat sediments. The dimension of each plot was 2.0 m (W) x 2.5 m (L) x 0.4 m (D) with sand median grain size of 0.95 mm. Clams were added to one side of the sediment corresponding to about 1000 clams.m -2 on July 26, 2002. Seawater was pumped from about 200 m far from seashore and was flowed to the mesocosms. The tide of seawater in the mesocosms was synchronized to water level in the natural condition. Sampling methods Pore water qualities, i.e. dissolved organic carbon (DOC), and ammonium nitrogen (NH 4 -N), in all plots were monitored during 24 hours to investigate the effect of clams on the changes of DOC and NH 4 -N concentrations in the sediments. Pore water sediment also used for molecular weight distribution analysis. About 20 ml of pore water for each analysis was taken from each plot of sediment by using peristaltic pump to suck water from capillary tube equipped with double layers of filter paper with 1.2 µm pore size (GF/C filter, Whatman). Sediment cores (n=2) were collected from 0-10 cm depth of each plot using an acrylic tube (5 cm i.d. x 50 cm long) and immediately used for biodegradability experiment. Sediment core sample also collected from 0-2 and 9-11 cm depth of sediment with (A1; A2) and without (B1; B2) clams, in order to evaluated NO 3 -N removal capacity and quinone analysis. A replaceable ring of acrylic tube with 4 cm i.d. and 2 cm thick was used to collect this sample. Sediment samples for NO 3 -N removal capacity experiment were used immediately after sampling, while the samples for quinone were stored at –20 o C until using for the analysis. Journal of Water and Environment Technology, Vol.2, No.2, 2004 - 85 - Laboratory experiment: experimental set up and procedure Biodegradability of organic carbon in the sediment was analyzed by using modified OECD screening test No. 301E. 5 gram of sediment was extracted with 60 ml seawater by using warring blender (3000 rpm, 10 min). The extract water was filtered with 0.3 µm filter (GF-75, Advantec). 40 ml of filtered extract as a test material was filled-in to 200 ml of Erlenmeyer flask. Seed and mineral solutions were also added. The flasks were shaken and incubated in the dark condition at 22 ± 2 o C. Decomposition was followed by DOC analysis at frequent interval over a 28-day period. The procedure was checked by means of a standard. A control with inoculation, but without either test material or standard, was run in parallel for the determination of DOC blank. NO 3 -N removal capacities were investigated in batch laboratory scale. Tidal flat sediment with 4 cm diameter and 2 cm thick was putted in 100 ml beaker glass. Seawater was filtered with 0.3 µm filter (GF-75, Advantec) to avoid from impurities. Sodium nitrate (NaNO 3 ) was used to enrich nitrate concentration in the filtered seawater. The initial NO 3 -N concentration was setup more than 4 mg N/l. The enriched seawater was purged with nitrogen gas for more than 15 min to make anaerobic condition. 60 ml of the treated seawater was added gently to each beaker glass and was incubated at 30 o C. The beaker glass was covered with parafilm to prevent oxygen transfer from air to liquid phase. Samples were taken every 3 hours during 12 hours. The removal rate (F) was calculated as follow: F = (C 0 -C t ).V/(W.t), where C 0 -C t = the differences between NO 3 -N concentrations at initial and at t time conditions; V=volume of seawater; W= sediment weight; and t= time. Analytical methods The DOC and NH 4 -N concentrations of pore water were measured with TOC analyzer (TOC- 5000A, Shimadzu) and automatic water analyzer (AACS-III, Bran+Luebbee), respectively. T-test statistical analysis was performed to distinguish the differences between pore water quality in the sediment with and without clams. NO 3 -N was analyzed using ion chromatography analyzer (DX- 120, DIONEX) equipped with Ion Pac column AS14 4-mm (10–32) (DIONEX) and a suppressor of ASRS-ULTRA 4-mm (DIONEX). Molecular weight distribution was analyzed by using gel permeation chromatography (GPC) analyzer equipped with a refractive index detector (RID-10A, Shimadzu) and Ultrahydrogel column 7.8 x 300 mm (Japan Waters Ltd.). Pore water was filtered with 0.45 µm cellulose acetate filter papers (Millipore), desalinated with Sephadex G-25 Medium column (Amersham Biosciences), and concentrated using freeze dryer before molecular weight distribution analysis. Approximate calibration of the column was accomplished by using poly- ethylene glycol as a standard. Microbial quinones in tidal flat sediment were analyzed using the method previously described (Hu, et al., 1999). Quinones were firstly extracted from the sediment using a mixture of chloroform-methanol and then re-extracted into hexane. Menaquinones and ubiquinones contained in the crude extract were separated and purified using Sep-Pak ® Plus Silica. The type and concentration of the quinones were determined using a HPLC equipped with an ODS column (Zorbax-ODS, 4.6(I.D.) x 250mm, Shimadzu-Dupont) and a photodiode array detector (SPD-M10A, Shimadzu Co., Japan). In this paper, the abbreviations of the type of quinone are ubiquinone: UQ, menaquinone: MK, plastoquinone: PQ and vitamin K1: K1. RESULTS AND DISCUSSION Diurnal variation of DOC concentration The diurnal variation of DOC concentration in pore water of tidal flat sediments with and without clams is shown in Fig. 1. Concentration of DOC in tidal sediment with clams was about 1.1-2.1 mg/l higher than that in the sediment without clams. Also, the DOC concentration increased with decreasing water level. This could be related with the activity of clams to consumed POM and excreted their fecal matters as a source of DOC. Pfeiffer et al. (1999) reported that most bivalves Journal of Water and Environment Technology, Vol.2, No.2, 2004 - 86 - Figure 1. The diurnal variation of water level, DO and DOC concentration in pore water of tidal flat sediments with and without clams were efficient at removing particle between 3-10 µm and total suspended solid removal rate of clams were about 0.4-4.9 (mg l -1 TSS g -1 clams day -1 ), depend on shell size. The difference between DOC concentration in the sediment with and without clams was not so high. This strongly suggests due to the low concentration of particulate organic carbon (POC) in the seawater. POC concentration in the seawater was only about 2.3-4.9 mg/l. Also, DOC production from clams excretion activities were consumed rapidly by microorganism in the sediment. Sempĕrĕ et al. (2000) reported that DOC was continuously released from POC and followed by a rapid cycling of DOC during particle decomposition. Whenever DO concentration increased, it was expected that the excretion activity of clams has increased. During emersion, the penetration of oxygen into sediments may increase (Durand et al., 2002; Kuwae et al., 2003) and will lead the production of DOC due to clam excretions activities. In addition, at low water level in the day (noon), light intensity reached to the highest level. The activity of photosynthetic bacteria might be stimulated to produce oxygen. DO concentration of overlying water at noon was about 7 mg/l, while at night was only about 5 mg/l (Fig.1). Due to the higher concentration of DO, DOC productions from clam activities also higher at low water level in the noon. Diurnal variation of NH 4 -N concentrations Fig. 2 shows that the concentration of NH 4 -N in tidal sediment with clams was higher than that in the sediment without clams. The concentration of NH 4 -N in this sediment was about 54% of dissolved total nitrogen (DTN). This result is agreed fairly well with the previous research. Zhu et al. (1999) has reported that the majority (60%) of the excreted nitrogen was in the form total ammonia nitrogen (TAN). The excretion rates of T. philippinarum were about 1.5-46.1 mg (kg.clam) -1 day -1 and 4.8-131.0 mg (kg.clam) -1 day -1 for TAN and TKN, respectively, indicating that NH 4 -N is the primary product of clam excretions activity. NH 4 -N concentration was also higher at low water level. As described in the previous section, during emersion and low water level DO concentration in tidal sediment was expected to increase and stimulate clams excretion activity. The diurnal variation of pore water quality indicated that clams could be used to improve the transformation of organic matters and nutrient in tidal flat sediment. 0 20 40 60 80 100 Water level (cm) 3 4 5 6 7 8 DO (mg/l) Wat er le vel D.O. 0 2 4 6 8 10 12 10:00 14:00 18:00 22:00 2:00 6:00 10:00 Without clams With clams DOC (mg/l) s ampling time Journal of Water and Environment Technology, Vol.2, No.2, 2004 - 87 - Figure 2. The diurnal variation of NH 4 -N concentration in pore water of tidal flat sediments with and without clams Biodegradability and molecular weight distribution of organic matter Figure 3 shows that more than 80% of DOC from the sediment with clams was removed during biodegradability test (within 28 days), while in the sediment without clams DOC removal was only about 40%. This indicated that clams promoted the production of readily biodegradable substances in tidal flat sediment. Clams consume particulate organic matters, digest that compounds in their body, and excrete their feces as sources of readily biodegradable substances. In the case of without clams, particulate organic matters degrade by microorganism through hydrolysis process. Degradation rate of the hydrolysis process is slower than degradation through clam activities. Therefore, DOC concentration and biodegradability of organic matters in the sediment with clams were higher than that in the sediment without clams. Figure 3. Biodegradability of organic matters in tidal flat sediment with and without clams Degradation of particulate organic matters by clams also affected molecular weight distribution in the pore water sediment. Figure 4 shows that clam decreased the molecular weight distribution of organic matters in the pore water sediment. In the sediment without clams, molecular weight of organic matters was distributed from 0.7x10 2 to 1.2x10 6 , while in the sediment with clams the molecular weight was distributed from 0.7x10 2 to 1.7x10 5 . The peak area of organic compounds with molecular weight about 1.7x10 5 in the sediment with clams was much higher than that in the sediment without clams. In contrast, in the sediment without clams, the amount of organic 0 1 2 3 13:00 20:00 2:00 8:00 sampling time NH 4 -N (mg/l) 0 20 40 60 80 100 water level (cm) with clam without clam water Level 0 20 40 60 80 100 0 7 14 21 28 Day DOC removal (%) without clam with clam Journal of Water and Environment Technology, Vol.2, No.2, 2004 - 88 - compounds with molecular weight about 1.2x10 6 was very low. This could be related with analytical method for determined molecular weight distribution. Prior to analysis, all samples were filtered using 0.45 µm cellulose acetate filter papers (Millipore). It means, the pore water only contain DOC and colloidal organic carbon (COC) (Sempĕrĕ et al., 2000) or without particulate organic matters. Becouse of this limitation, peak area of organic matters in the pore water of sediment with clams was higher than that in the sediment without clams. The ability of clams to degrade particulate organic matters increased the concentration of soluble materials (DOC and others compounds) with lower molecular weight. In the case of the sediment without clams, the degradation of particulate organic matters are likely due to an ectoenzymatic activity of attached bacteria which renders the particles soluble through macromolecular hydrolysis and produces dissolved organic compounds and small molecules which are subsequently taken up by attached and free-living bacteria (Karner and Herndl, 1992; Smith et al., 1992; Sempĕrĕ et al., 2000). Figure 4 also shows that at 15 cm depth, the molecular weight distributions in the pore water of both sediments were relatively similar. This strongly suggests that the activity of clams in about 15 cm depth might be decreased significantly due to the limitation of DO in the sediment. Figure 4. Molecular weight distribution of organic matters in tidal flat sediment with and without clams Nitrate removal capacity Figure 5 shows that high concentration of microorganism in the sediment has correlation with high removal rate of NO 3 -N from the sediment, especially in the sediment with clams. The increases of carbon and nutrient concentration with higher biodegradability promoted the microbial growth in the sediment with clams, indicated by higher concentration of quinone. Quinone content in the sediment with clams (A) was higher than that in the sediment without clams (B). Also, quinone content in 0-2 cm depth of sediment was higher than that in 9-11 cm depth of sediment. This could with clams 5cm depth 0 1000 2000 3000 4000 5000 Intensity (mV) 10 5 10 4 10 3 10 2 176343 10 5 10 4 10 3 10 2 409338 with clams 15 cm depth without clams 5 cm depth 0 1000 2000 3000 4000 5000 0 102030405060 Retention time (min) Intensity ( µ V) 1184816 0 102030405060 Retention time (min) 479364 without clams 15 cm depth Molecular weight Intensity (µV) Intensity (µV) Retention time (min) Retention time (min) Journal of Water and Environment Technology, Vol.2, No.2, 2004 - 89 - 0 0.2 0.4 0.6 A1 B1 A2 B2 NO3-N removal rate (mg/kg. sediment.h) 0 0.4 0.8 1.2 Quinone content ( µ mol/kg. dry sediment) PQ+VK1 MK UQ Removal rate Figure 5. NO 3 -N removal rate in tidal flat sediment with (A) and without (B) clams. Index 1 and 2 indicated 0-2 cm and 9-11 cm depth of sediment, respectively be related with DO and substrate limitation in the deeper sediment. High concentration of microorganism in the sediment with clams was followed by higher NO 3 -N removal rate. While, in the case of sediment without clams, higher concentration of microorganism in 0-2cm depth of sediment was not followed by higher NO 3 -N removal rate proportionally. This indicated that microbial communities in the sediment with and without clams are different. NH 4 -N concentration in tidal flat sediment with clams was about 2 times higher than that in the sediment without clams. This condition was suitable for the growth of nitrifying bacteria and increase nitrification process in tidal flat sediment with clams. Usui et al. (2001) reported that the availability of NH 4 + is one of major factors regulating nitrification in the coastal marine sediment. High activity of nitrifying bacteria produced higher concentration of NO 3 -N. Also, the existence of clams consumed DO and decreased DO concentration in the sediment. Low oxygen concentration or anaerobic condition is suitable for the growth of denitrifying bacteria (Usui et al., 2001). NO 3 -N removal capacity in 2 cm of the top layer of tidal flat sediment with clams was about 3 times higher than that of tidal flat sediment without clams. High concentration of denitrifying bacteria and low oxygen concentration were suitable condition for NO 3 -N removal from tidal flat sediment. High concentration of photosynthetic (photoheterotrophic) bacteria, which is indicated by PQ and VK1, also increased NO 3 -N removal rate through NO 3 -N utilization as a terminal electron acceptor in their metabolism (Lester and Birkett, 1999). The increased of sediment depth decreased NO 3 -N removal capacity. This could be related with the decreasing of microbial concentration and an absence of photoheterotrophic bacteria in the lower sediment. CONCLUSIONS Clams appear to be a suitable organism to accelerate the decomposition of organic matters in tidal flat sediment. Clams activities increased DOC, NH 4 -N concentration and biodegradability of organic matters in tidal flat sediment. Also, clams decreased molecular weight distribution of organic matters in the pore water sediment. The symbiotic relationship between clams and microorganisms accelerated decomposition of organic matters in tidal flat area and increased nitrate removal capacity of tidal flat sediment. Results of this study are useful to develop a method of tidal flat conservation. Journal of Water and Environment Technology, Vol.2, No.2, 2004 - 90 - REFERENCES Durand, F. Peters, L.D. and Livingstone, D.R. (2002). Effect of intertidal compared to subtidal exposure on the uptake, loss, and oxidative toxicity of water born benzo[a]pyrene in the mantle and whole tissues of mussel Mytilus edulis, L. Marine Environmental Research, Vol. 54, 271- 274. Graco, M., Farias, L., Molina, V., Gutierrez, D. and Nielsen, P.L. (2001). Massive developments of microbial mats following phytoplankton blooms in a naturally eutrophic bay: implication for nitrogen cycling. Limnology and Oceanography, Vol. 46, No. 4, 821-832. Hasanudin, U., Shimada, K., Kunihiro, T., Fujie, K. and Suzuki, T. (2004). The effect of clams on carbon and nitrate removal potential of tidal flat sediment. Japan Society on Water Environment (JSWE) annual conference, March 17-19, Sapporo, Japan . p. 163. Hu, H. Y., Fujie, K., and Urano, K. (1999). Development of a novel solid phase extraction method for the analysis of bacterial quinines in activated sludge with a higher reliability. Journal of Bioscience and Bioengineering, Vol. 87, No. 3, 378-382. Karner, M. and Herndl G. J. (1992). Extracellular enzyme activity and secondary production in free living and marine-snow-associated bacteria. Marine Biology, Vol.113, 341-347. Kuwae, T., Kibe, E. and Nakamura, Y. (2003). Effect of emersion and immersion on the porewater nutrient dynamics of an intertidal sandflat in Tokyo Bay. Estuarine Coastal and Shelf Science, Vol. 57, 929-940. Lester, J.N. and Birkett, J.W. (1999). Microbiology and chemistry for environmental scientists and engineers. (2 nd edition). E & FN Spon, London. Magalhaes, M. C., Bordalo, A. A. and Wiebe, J.W. (2003). Intertidal biofilms on rocky substratum can play a major role in estuarine carbon and nutrient dynamics. Marine Ecology Progress Series, Vol. 258, 275-281. Magni, P., Montani, S., Takada, C. and Tsutsumi, H. (2000). Temporal scaling and relevance of bivalve nutrient excretion on a tidal flat of the Seto Inland Sea, Japan. Marine Ecology Progress Series, Vol. 198, 139-155. Ogilvie, B., Nedwell, D.B., Harrison, R.M., Robinson, A. and Sage, A. (1997). High nitrate, muddy estuaries as nitrogen sinks: the nitrogen budget of the River Colne estuary (United Kingdom). Marine Ecology Progress Series, Vol. 150, 217-228. Okabe, S., Matsuda, T., Satoh, H., Itoh, T. and Watanabe, Y. (1998). Sulfate reduction and sulfide oxidation in aerobic mixed population biofilms. Water Science and Technology, Vol. 37, No. 4-5, 131-138. Pfeiffer, J. T., Lawson, B. T. and Rusch, A. K. (1999). Northern quahog, Mercenaria mercenaria, seed clam waste characterization study: precursor to a recirculating culture system design. Aquaculture Engineering, Vol. 20, 149-161. Sempĕrĕ, R., Yoro, C. S., Wambeke, F. V. and Charriere. (2000). Microbial decomposition of large organic particles in the northwestern Mediterranean Sea: an experimental approach. Marine Ecology Progress Series, Vol. 198, 61-72. Smith, D.C., Simon, M., Alldredge, A. and Azam, F. (1992). Intense hydrolytic enzyme activity on marine aggregates and implications for rapid particles dissolution. Nature, Vol. 359, 139-142. Ueda, N., Tsutsumi, H., Yamada, M. and Hanamoto, K. (2000). Impacts of oxygen-deficient water on the macrobenthic fauna of Dokai Bay and on adjacent intertidal flats, in Kitakyushu, Japan. Marine Pollution Bulletin, Vol. 40, 906-913. Usui, T., Koike, I. and Ogura, N. (2001). N 2 O production, nitrification and denitrification in an estuarine sediment. Estuarine, Coastal and Shelf Science, Vol. 52, 769-781. Zhu, S., Saucier, B., Durfey, J., Chen, S. and Dewey, B. (1999). Waste excretion characteristics of Manila clams (Tapes philippinarum) under different temperature conditions. Aquaculture Engineering, Vol. 20, 231-244. . Environment Technology, Vol .2, No .2, 20 04 - 89 - 0 0 .2 0.4 0.6 A1 B1 A2 B2 NO3-N removal rate (mg/kg. sediment.h) 0 0.4 0.8 1 .2 Quinone content ( µ mol/kg 2 3 13:00 20 :00 2: 00 8:00 sampling time NH 4 -N (mg/l) 0 20 40 60 80 100 water level (cm) with clam without clam water Level 0 20 40 60 80 100 0 7 14 21